Purification and partial characterisation of recombinant human hepcidin

Hepcidin is a liver-expressed antimicrobial and iron regulatory peptide. A number of studies have indicated that hepcidin is important for the correct regulation of body iron homeostasis. The aims of this study were to analyse the expression, trafficking and regulation of human hepcidin in an in vitro cell culture system. Human hepcidin was transfected into human embryonic kidney cells. Immunofluorescence and confocal microscopy analysis revealed that recombinant hepcidin localised to the Golgi complex. Recombinant hepcidin is secreted from the cell within 1 h of its synthesis. Recombinant hepcidin was purified from the cell culture medium using ion-exchange and metal-affinity chromatography and was active in antimicrobial assays. Amino-terminal sequence analysis of the secreted peptide revealed that it was the mature 25 amino acid form of hepcidin. Our results show that recombinant myc-His tagged human hepcidin was expressed, processed and secreted correctly and biologically active in antimicrobial assays. (C) 2005 Elsevier SAS. All rights reserved.

Quantitative analysis of MC1R gene expression in human skin cell cultures

To address the issue of melanocortin-1 receptor (MC1R) expression in non-melanocytic cells, we have quantitatively evaluated the relative expression levels of both MC1R mRNA and protein in a subset of different cell types. Using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) at high cycle numbers, we detected MC1R mRNA in all cell types examined, including human embryonic kidney-293 (HEK 293) cells, a cell type widely used as a negative control in melanocortin expression studies. Quantitative real-time PCR revealed the highest levels of MC1R transcripts were in melanocytic cells, whereas the keratinocyte and fibroblast cell cultures examined had only a low level of expression, similar to that of HEK 293 cells. Antibody mediated detection of MC1R protein in membrane extracts demonstrated exogenous receptor in MC1R transfected cell lines, as well as endogenous MC1R in melanoma cells. However, radioligand binding procedures were required to detect MC1R protein of normal human melanocytes and no surface expression of MC1R was detected in any of the non-melanocytic cells examined. This was consistent with their low level of mRNA, and suggests that, if present, the levels of surface receptor are significantly lower than that in melanocytes. The capacity of such limited levels of MC1R protein to influence non-melanocytic skin cell biology would likely be severely compromised. Indeed, the MC1R agonist [NIe(4), D-Phe(7)] alpha-melanocyte stimulating hormone (NDP-MSH) was unable to elevate intracellular cyclic adenosine monophosphate (cAMP) levels in the keratinocyte and fibroblast cells examined, whereas a robust increase was elicited in melanocytes. Although there are a variety of cell types with detectable MC1R mRNA, the expression of physiologically significant levels of the receptor may be more restricted than the current literature indicates, and within epidermal tissue may be limited to the melanocyte

Testicular germ cell tumors exhibit evidence of hormone dependence

The aim of this investigation was to test the hypothesis that testicular germ cell tumors (TGCTs) are hormone-dependent cancers. Human TGCT cells were implanted in the left testis of male severe combined immunodeficient mice receiving either no treatment or hormone manipulation treatment [blockade of gonadotropin-releasing hormone secretion and/or signaling using leuprolide or leuprolide plus exogenous testosterone]. Real-time RT-PCR analysis was used to determine the expression profiles of hormone pathway-associated genes. Tumor burden was significantly smaller in mice receiving both leuprolide and testosterone. Real-time RTPCR analysis of follicle-stimulating hormone (FSH) receptor, luteinizing hormone (LH) receptor and P450 aromatase revealed changes in expression in normal testis tissue related to presence of xenograft tumors and manipulation of hormone levels but a complete absence of expression of these genes in tumor cells themselves. This was confirmed in human specimens of TGCT. Reduced TGCT growth in vivo was associated with significant downregulation of LH receptor and P450 aromatase expression in normal testes. In conclusion, manipulation of hormone levels influenced the growth of TGCT in vivo, while the presence of xenografted tumors influenced the expression of hormone-related genes in otherwise untreated animals. Human TGCTs, both in the animal model and in clinical specimens, appear not to express receptors for FSH or LH. Similarly, expression of the P450 aromatase gene is absent in TGCTs. Impaired estrogen synthesis and/or signaling may be at least partly responsible for inhibition of TGCT growth in the animal model. (c) 2005 Wiley-Liss, Inc.

The effects of ethanol on PPARS in MCF-7 human breast cancer cells

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors involved in various metabolic diseases. In the liver, PPARα is involved in alcohol metabolism and may lead to the development of alcoholic fatty liver and other alcohol mediated liver injuries. PPARβ modulation by ethanol induces abnormal myelin production by oligodendrocytes. PPARα and PPARβ are PPAR isoforms expressed in the human breast cell lines. Epidemiological studies show a positive correlation between alcohol intake and breast cancer risk, however, the molecular mechanisms involved are unclear. We hypothesized that ethanol would affect the expression and transactivation of human PPAR isoforms in estrogen receptor (ER) positive and ER negative breast cancer cells. Using real time RT-PCR we looked at the transcription of PPAR isoforms in the presence of increasing concentrations of ethanol and saw isoform and time dependent specific effects. Gene reporter assays enabled us to ascertain the effects of ethanol on ligand-mediated activation of human PPARα and PPARβ at concentrations equivalent to both moderate and chronic alcohol consumption. Ethanol differentially blocked the ligand-mediated activation of both PPARα and PPARβ. Since PPARα and PPARβ are involved in the differentiation and proliferation of breast cancer cells, PPARs may be a possible mechanism involved in the effect of ethanol in breast cancer.